Cycloxygenase (COX), or prostaglandin H2 (PGH2) synthase is the key enzyme able to catalyze the formation of prostaglandins from arachidonic acid. PGH2, the product of COX, is the common precursor for the biosynthesis of prostaglandins, prostacyclins and thromboxanes. COX is the well known target of nonsteroidal anti-inflammatory drugs (NSAIDs), which have been used for about one century as first line therapy for relieving inflammation and pain associated with a number of arthritic conditions. Prostaglandins (PGD2, PGI2, PGE2, PGF2α) have various effects on mammalian physiology, in particular prostaglandin E2 (PGE2), which is the predominant eicosanoid detected in inflammation conditions, is an important mediator of pain, fever and other symptoms associated with inflammation. Accordingly, inhibition of the biosynthesis of prostaglandins has been a target for the therapeutic treatment of pain and inflammatory conditions for years. Main adverse-effect associated with the chronic usage of NSAIDs, is for a great part of the population, gastrointestinal irritation which can give rise to life threatening lesions and ulceration if the therapy is not interrupted. Another rather common adverse effect of NSAIDs is renal toxicity. An alternative to NSAIDs is the use of corticosteroids, however also in this case chronic use can result in severe side effects.
In the early 1990s it was demonstrated that COX exists as two distinct isoforms that catalyze the same reaction but differ in terms of regulation; in particular it was shown how COX-1 is constitutively expressed as a house keeping enzyme in almost all tissues, and is responsible for those physiological functions such as for example cytoprotection of the gastrointestinal tract, platelet aggregation, vascular tone. On the other hand, COX-2, the second isoform, was identified as an inducible enzyme, highly expressed in response to inter-leukin-1β (IL-1β) and other inflammatory stimuli. Thus, COX-2 was proposed as responsible for the production of prostaglandins associated with pain and inflammatory conditions. The expression of COX-2 is indeed regulated by a broad spectrum of mediators involved in inflammation in the most of tissues. Whereas lipopolysaccharide and proinflammatory cytokines (IL-1β, TNFα) can induce COX-2, glucocorticosteroids and interleukins-4, -13 and -10 inhibit the expression of this enzyme in response to an anti-inflammatory stimulus. Thus, several line of evidence supported the selective inhibition of COX-2 as a potentially powerful new mechanism for the treatment of the inflammatory related diseases and relief of acute pain, with a lower incidence of gastrointestinal-related adverse events compared with non-selective NSAIDs.
This led to a tremendous medicinal chemistry effort which gave rise in a few years to a plethora of compounds (including Rofecoxib, Celecoxib, Valdecoxib, Parecoxib and later Etoricoxib and Lumiracoxib) (Figure 1) endowed with different COX-1/COX-2 selectivity, and different pharmacokinetic and toxicological profiles.
Clinical trials with these compounds largely confirmed the effectiveness of the approach in the treatment of inflammatory disorders such as arthritis and for the management of acute pains in adults, as well as confirmed the expected safety of these drugs as far as gastrointestinal damages are concerned. Celecoxib and Rofecoxib were shown, in strict clinical trials, to cause a significantly lower incidence of upper gastrointestinal adverse effects (perforations and ulcers) in comparison to classical NSAIDs.
Whereas in the early 1990s COX-2 was regarded as an enzyme only connected with inflammatory diseases and pain, in the following years its involvement in other pathologies turned out.
Epidemiological studies highlighted at the end of the 1990's how the risk of developing Alzheimer's disease (AD) was significantly reduced among users of NSAIDs. Recently, several lines of evidence established the role of COX-2 in AD (Arch. Gerontol. Geriatr., 2001, 33, 13-28). It was shown how in AD COX-2 is up regulated in brain areas related to memory (hippocampus, cortex), with the amount of COX-2 correlating with the amount of deposition of β-amyloid protein in the neuritic plaques. Several works demonstrated how this correlation between COX-2 activation and deposition of β-amyloid is supported by a well defined mechanism. In a transgenic mouse model of Alzheimer's disease it was shown that COX is influencing Amyloid-β peptide generation through a mechanism that involve PGE2 mediated potentiation of γ-secretase activity (J. Biol. Chem., 2003, 278, 51, 50970). In addition it was reported that in astrocytes β-amyloid peptide 25-35 induces COX-2 mRNA and protein synthesis with subsequent release of PGE2 by the astrocyte. This could give rise to a self-amplifying loop where PGE2 is potentiating Amyloid-β peptide generation, which in turn potentiate PGE2 production. Elevation of COX-2 expression in hippocampal neurons during the early phase of AD (mild dementia) is considered to favour the later neurodegenerative process. Further evidences suggest that COX-2 derived prostanoids potentiate glutamate excitotoxicity, thereby accelerating neurodegeneration. Thus, accumulating findings are showing how COX-2 inhibitors can prevent AD and even counteract the progression of the disease (MG Giovannini, Experimental brain inflammation and neurode-generation as model of Alzheimer's disease: protective effects of selective COX-2 inhibitors).
Many brain disorders such as Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington disease, HIV induced dementia and head trauma are associated with inflammation. Microglia and astrocytes act as immune cells in the inflamed brain, and especially microglia contribute to the onset of inflammation in many brain disease by producing proinflammatory mediators, among them prostaglandins.
A microglia reaction associated with up-regulation of COX-2 along with iNOS has been suggested to play an important role in dopaminergic neuron loss in PD (J. of Neuroinflammation 2004, 1:6; J. of Neuroinflammation 2006, 3:6), and selective COX-2 inhibition has been shown to prevent progressive degeneration in a rat model of Parkinson's disease.
Recent studies pointed out the presence of inflammatory markers in affected neural tissues of ALS patients, and macrophages in the ALS spinal cord highlighted strong expression of COX-2. Further studies suggested that COX-2 could promote motor neuron loss in rodent models of ALS while COX-2 inhibitors can significantly delay the onset of motor dysfunction in a transgenic mouse model of ALS (FASEB J., 2003, 17, 6, 725).
Human immunodeficiency virus type-1 (HIV-1)-associated dementia (HAD) is a neurodegenerative disease characterized by HIV infection and replication in brain tissue. HIV infected monocytes overexpress COX-2 as demonstrated both in vitro and in vivo models (J. Leukoc. Biol., 2000, 68, 3 423). Also in this case overproduction of inflammatory prostaglandins trigger or contribute to the triggering of those inflammation-mediated neurodegenerative processes which in turn give rise to neuronal loss (Current Drug Targets Inflamm. Allergy, 2005, 3, 335).
Among major targets for COX-2 inhibition, COX-2 expression by vulnerable neurons and by microglia which in turn give rise to the neuroinflammatory response (J. Neurotrauma, 2002, 19, 1). have been identified for a potential treatment of brain injury. Evidence suggests that using an appropriate treatment paradigm, COX-2 inhibition can impact these three targets and thus will be a useful, ameliorative adjunct in the treatment of most forms of brain injuries.
The capacity of NASIDs, such as Aspirin and Sulindac, to reduce colorectal cancer mortality was highlighted by clinical studies during the 1980s. Later the clinical efficacy of COX-2 inhibitors in cancer chemoprevention was demonstrated by randomised studies in patients with a precancerous condition. An involvement of COX-2 in several forms of solid tumors is supported by its over-expression in gastric, hepatic, esophageal, pancreatic, head and neck, colorectal, breast, bladder, skin and lung cancers when compared with non-malignant controls.
In five large-size clinical trials involving the COX-2 inhibitors: Celecoxib, Rofecoxib, Valdecoxib was highlighted an increased incidence of myocardial infarction and stroke. In September 2004 Rofecoxib was voluntarily withdrawn from the market for increased cardiovascular risk and the same happened in April 2005, with Valdecoxib. Though, incidence of adverse cardiovascular events with Rofecoxib is quite evident, especially considering the results of the VIGOR trial (Vioxx Gastrointestinal Outcomes Research; P. Juni, Risk of cardiovascular events and Rofecoxib: cumulative meta-analysis, Lancet, 2004, 364, 2021), where there was a five-fold increase in the incidence of acute myocardial infarction in the Rofecoxib arm of the trial when compared with the Naproxen arm, clinical results of trials dealing with the same adverse-effect didn't highlight any significant difference when Celecoxib was compared to classical NSAIDs. In order to explain this unexpected cardiovascular toxicity, it was speculated that selective COX-2 inhibitors may block the production of prostacyclin (antithrombotic) and leave thromboxane (prothrombotic) generation unaffected. This explanation, which could suggest the increased cardiovascular risk is a feature of the whole class of selective COX-2 inhibitors, is supported by the recent findings that COX-2 is constitutively expressed by some tissues, such as vascular endothelium and kidney. According to this explanation, selective COX-2 inhibitors cannot inhibit COX-1, constitutive within the platelets and associated with the production of thromboxane A2 (TXA2; a potent inducer of platelet aggregation and vasoconstriction), but can inhibit the endothelial COX-2 which is producing prostacyclin, that inhibits platelet aggregation and give rise to potent vascular smooth muscle dilation. This selective inhibition of the vasodilatation/anti-aggregation promoter without concomitant inhibition of the vasoconstrictor/pro-aggregant stimulus TXA2 (as usually happens with non selective NSAIDs) should explain the increased evidence of cardiovascular risk when comparing selective COX-2 inhibitors with classical NSAIDs. The same explanation could also address the potential renal toxicity attributed to the class of COX-2 inhibitors.
This hypothesis is well matching with the first clinical data where the less selective Celecoxib displayed no cardiovascular adverse event, however is not matching with a second set of clinical data coming from a study aimed at establishing whether Celecoxib could treat AD, where incidence of heart attack was four times higher in the treated group in comparison to placebo (JMC, 2005, 2251-2257).
Also in contradiction with this hypothesis is the fact that for the highly COX-2 selective inhibitors Etoricoxib and Lumiracoxib, the evidences for cardiovascular adverse events, in specifically designed clinical trials, seems to be more ambiguous in comparison to the ones produced for Rofecoxib.
Looking at the overall available body of data concerning the relationships between selective COX-2 inhibitors and cardiovascular events at least conflicting evidence could be recognized. The cardiovascular risks of COX-2 inhibitors appear heterogeneous, influenced not only by the drug class, but also by the individual structure of the drug, and by the dosage (Expt. Opin. Drug Saf. 2005, 4, 6, 1005).
Taking into account the structures of the best known inhibitors (Figure 1) it could be recognized that Rofecoxib, Celecoxib and Valdecoxib closely share the diaryl substituted heterocyclic moiety while Etoricoxib for some extent and for a larger extent Lumiracoxib are progressively differentiating from this common simple scaffold.
Diversity in the chemical structure of COX-2 inhibitors not only could account for differences in the ADME profiles (i.e.: Celecoxib which is extremely high lipophilic is sequestered in body fat while Lumiracoxib, due to the acetic moiety, reaches higher circulating concentration) which in turn may also reflect in a different cardiovascular impact, but also can influence pharmacological responses not mediated by COX-2 but due to other mechanisms.
For example, even in the quite homogeneous class of the diaryl-substituted heterocycles Celecoxib and Valdecoxib sharing the arylsulphonamide group with many carbonic anhydrase inhibitors, are able to reduce intraocular pressure (not COX-2 dependent effect) in glaucomatous rabbits, while Rofecoxib which has the methylsulphonyl group instead of the sulphonamide group, had no effect. Conversely, the sulphone group in the structure of Rofecoxib increases susceptibility of low-density lipoprotein to oxidative modifications, while Celecoxib had no effect (J. Med. Chem., 2005, 48, 2255). Diaryl-substituted heterocycles are suitable scaffolds for interaction with the ATP site of protein kinases. For example, Celecoxib induces apoptosis by a COX-2 independent mechanism (Cancer Research, 2004, 64, 4309) by blocking 3-phosphoinositide-dependent protein kinase-1 (PDK-1) which in turn activate Akt (Protein Kinase B) a kinase involved in cell cycling. These examples highlight how many different effects, not COX-2 dependent, can be found even in few related structures.
The above issues along with the previously mentioned conflicting body of clinical data regarding COX-2 inhibitors and adverse cardiac event, suggest that this side effect could be completely or in part structure related. Structural issues can also reflect in unfavourable ADME profiles and/or action through other mechanisms which could give rise to an higher or lower cardiovascular toxicity as a whole outcome.
These considerations indicated that there is still need for structurally diversified COX-2 inhibitors possibly able to overcome the safety issues highlighted with the first and second generation of this group of highly effective and useful drugs.

Looking at the structures in Figure 1, with exclusion of Etoricoxib and Lumiracoxib it could be recognized how diaryl-substituted penta-atomic heterocycles could be particularly well shaped for originating potent and selective COX-2 inhibitors. However, it was also above discussed how this pattern can be in addition well suited for interaction with the ATP site of kinases, for instance as reported for Celecoxib and PDK-1. In addition, the 1,2-diaryl substitution pattern can be recognized in a number of penta-atomic heterocycles, particularly imidazoles and pyrazoles, for example in the class of MAP kinase inhibitors (Current Topics in Medicinal Chemistry, 2002, 2, 1011; Bioorganic Medicinal Chemistry Letters, 2004, 3581). The too high lipophilic character of Celecoxib, which reflected in a drug with not completely favourable ADME characteristics, also suggested to avoid too lipophilic scaffolds.
In order to decrease the possibility of unwanted potentially not safe interaction with other targets (es. kinases) while selecting scaffolds with not too high lipophilicity, in order to optimize ADME characteristics, we preferred the pyrrole nucleus as the core heterocycle. The pyrrole nucleus as well as suitably substitutes pyrroles are moieties present in several natural and safe substances. This heterocycle is not particularly unstable from a metabolical standpoint, does not give rise to reactive intermediates by metabolic activation, is endowed of a suitable hydrophilicity. As drugs pyrroles have been described for various uses including treatment of inflammation (Il Farmaco, 1984, 756-80; where 1,5-diaryl-3-methyl-pyrroles are discussed), pain pharmacological treatment (DE 1972-2261965; where 1-aryl-2,5-dimethyl-3-aminomethyl-pyrroles are discussed as analgesics and antipyretic, for cardiovascular therapeutic treatment (EP 0323841, where 1-methylaryl-5-alkyl-2-substituted-pyrroles along with pyrazole and triazole are described as angiotensin II antagonists). 1,5-diaryl-2-methyl-3-aminomethyl-pyrroles have been described as antibacterial agents and anti-candida agents (F. Cerreto, Eur. J. Med. Chem. 1992, 27, 701; M. Scalzo, Il Farmaco, 1988, 43, 655). In the case of this pyrrole derivatives the presence of the aminomethyl group in position 3 is fundamental for the microbiological activity, while aromatic rings substitution pattern is modulating this activity. 1,5-Diaryl-pyrroles as selective COX-2 inhibitors have been described (WO98/25896); in this patent application, though the substitution of the aromatic rings in part is matching the corresponding one in this patent application, striking structural differences in the substitution of the pyrrole ring at position 2 and particularly at position 3 can be recognized between the two inventions. Synthetic preparation of the 1,2-diaryl-pyrroles has been reported by Stetter (H. Stetter, Angew. Chem. Int. Ed. Engl., 1976, 15, 639) and more recently by Khanna (I. K. Khanna, J. Med. Chem., 1997, 40, 1619).
The safety profile of Lumiracoxib along with its favourable ADME characteristics suggest how a proper functionalized side chain can modulate overall drug solubility and lipophilicity, as well as could limit undesirable interactions with possible targets other than COX-1/2. As the matter of fact, the first generation of COX-2 selective inhibitors is not represented by structures particularly functionalized, they are rather simple structures that if on one hand are able to achieve an high COX-1/COX-2 selectivity, on the other hand could potentially interact with several other targets.
Accordingly, increasing compound functionalization can be a means, for those cases where activity and COX-1/COX-2 selectivity is retained, to increase product safety by decreasing possible interactions with undesidered targets and/or properly modulating drug ADME characteristics.
1,5-Diarylpyrroles bearing side chains functionalized with carboxylate or related moieties have been reported previously. 1,5-Diarylpyrroles bearing in the position 2 the butanoic as well as the propanoic chains have been described (I. K. Khanna, mentioned above), these compounds however were found to be very week inhibitors of COX-2 both as esters and carboxylic acids independently upon the substitution pattern at the aromatic rings in positions-1 and -5. The presence of lipophilic substituents in the pyrrole position-3 give rise to potent COX-2 inhibitors when the substituent is an halogen atom or a trifluoromethylsulphonyl group. Introduction in position-3 of short side-chains such as an hydroxymethyl or a dimethylaminomethyl have been proved deleterious for COX-2 inhibitory activity (v.s.), while longer and more functionalized chains have not been yet reported.